The ultimate goal of this proposal is to understand catalysis by the HDV ribozyme. Ribozymes are ideal model systems for the vast number of non-protein coding RNAs found in all kingdoms of life. They have high biological and biotechnological relevance in their own right for their ability to process and regulate genetic information. Yet, two decades after their discovery, our understanding of ribozyme catalysis still pales compared to that of protein catalysis. A particularly intriguing and enigmatic example is the HDV ribozyme. It is the only ribozyme found in a human pathogen, the hepatitis delta virus (HDV), and has recently been discovered to also reside in the human genome. Multiple crystal structures exist that, together with detailed enzymatic studies, suggest a direct catalytic role of a specific nucleotide of the RNA chain, C75, as either general base or acid. During the previous funding cycle we discovered and characterized a conformational change that accompanies catalysis in solution and, as supported by subsequent crystallographic and molecular dynamics studies, appears to drive the catalyzed cleavage reaction forward. Most recently we have discovered that a ubiquitous U-turn motif is at the heart of this structural switch and exposes the cleavage site to the catalytic C75. This discovery links numerous, previously unconnected observations on the HDV ribozyme and offers the opportunity to fully characterize the complex relationship between RNA sequence, conformational change, folding free energy, and reaction chemistry, a first for any ribozyme. To take advantage of this opportunity and validate our methodology, we will employ an integrated set of biophysical and biochemical tools and will pursue the specific aims detailed below. Since the U-turn is one of the most common RNA structural motifs, our results promise to also have broad impact on our understanding of structural dynamics and function of the hammerhead ribozyme, tRNAs, HIV-1 genomic RNA, and GNRA tetraloops of large structured RNAs. In Specific Aim 1, we will experimentally measure the structural dynamics around the U-turn of the HDV ribozyme cleavage site by introducing (1) fluorophore pairs for single-molecule fluorescence resonance energy transfer (FRET) measurements of global conformational dynamics at the timescale of milliseconds and beyond;and (2) 13C,15N-labeled nucleotides as site-specific NMR probes to analyze local as well as global structural dynamics at the pico- to millisecond timescale. In Aim 2, we will solve the structure ensemble of the HDV ribozyme at atomic resolution and map its free energy landscape with advanced molecular dynamics simulations that will be guided by our experimental results from Aim1. In Aim 3, we will derive a full QM/MM description of HDV ribozyme catalysis based on the structural dynamics map available from Aim 1 and 2. Finally in Aim 4, we will rigorously test our model of catalysis by introducing specific chemical modifications into the ribozyme's U-turn, with the potential to improve catalytic efficiency of the naturally evolved HDV ribozyme wild-type.